Microvesicles (mvs) derived from adult stem cells for use in the therapeutic treatment of a tumor disease

ABSTRACT

The invention is in the field of therapeutic treatment of tumours. The inventors have found that microvesicles derived from adult stem cells exert a remarkable anti-tumour effect when administered to a patient affected by a tumour disease. Preferred microvesicles are derived from a bone marrow-mesenchymal stem cell, a glomerular mesenchymal stem cell or a non-oval liver stem cell.

The present invention relates to the therapeutic treatment of a tumourdisease.

Hematopoietic stem cells transplantation is known to exert tumorinhibitory effects in patients with solid tumor, as well as anti-tumoreffects in metastatic breast, kidney, ovarian, prostate and pancreaticcancer.

Human bone marrow mesenchymal stem cells (BM-MSCs) were demonstrate tocontribute to the repair of a wide variety of organs and tissues repairand experimental studies suggested that transplantation of MSCs may havea beneficial effect on functional and structural recovery in severalorgans including hearth, liver and kidney.

The stem cells microenvironment seems to play an essential role inpreventing carcinogenesis by providing signals to inhibit proliferationand to promote differentiation.

However, the use of stem cells in therapeutic indications is lessadvisable given that a potential tumorigenic risk of such stem celltherapy has been reported (Amariglio N et al. Donor-derived brain tumorfollowing neural stem cell transplantation in an ataxia telangiectasiapatient. PLoS Med. 2009 Feb. 17; 6(2))

Cell-derived microvesicles (MVs) are small vesicles released by cellsthat express the characteristic antigens of the cell from which theyoriginate and carry membrane and cytoplasmic constituents and have beendescribed as a new mechanism of cell communication. Recently, thepresent inventors demonstrated that microvesicles derived from humanendothelial progenitor cells (EPCs), BM-MSCs and hepatic liver stemcells may serve as a vehicle for transfer of genetic material (mRNA)that can reprogram target differentiated cells, as endothelial cells,tubular epithelial cells and hepatocytes (Deregibus M C et al.Endothelial progenitor cell derived microvesicles activate an angiogenicprogram in endothelial cells by a horizontal transfer of mRNA. Blood.2007 Oct. 1; 110(7):2440-8; Bruno S et al. Mesenchymal stem cell-derivedmicrovesicles protect against acute tubular injury. J Am Soc Nephrol.2009 May; 20(5): 1053-67; Herrera M B et al. Human liver stemcell-derived microvesicles accelerate hepatic regeneration inhepatectomized rats. J Cell Mol Med. 2009 Jul. 24) and contribute totissue regeneration and repair.

International patent application WO2009/087361 discloses producingdifferentiated cells by applying an inducer to a first population ofundifferentiated cells and then isolating microvescicles from thedifferentiated cells. See in that respect the first full paragraph onpage 30 of WO2009/087361, The first population of undifferentiated cellsis e.g. Bone marrow mesenchymal stem cells. However, WO2009/087361 doesnot disclose obtaining microvesicles directly from the undifferentiatedcells.

The present inventors have now found that microvesicles (MVs) derivedfrom adult stem cells, preferably from bone marrow or glomerularmesenchymal stem cells or from non-oval liver stem cells, showremarkable anti-tumour activities both in vitro and in vivo, therebyrepresenting an advantageous alternative over the corresponding wholestem cells for the therapeutic treatment of cancer. The anti-tumouractivity of microvesicles derived from adult stem cells according to thepresent invention was demonstrated in vitro by measuring the effect ofthe microvesicles on the proliferation and apoptosis of a variety ofhuman cancer cell lines as well as their effect on the formation ofcapillary-like structures. The anti-tumour activity of the microvesicleswas also confirmed in an in vivo mice model by measuring the effect ofMVs-treatment on tumour growth.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and B graphically show the incubation results of HepG2 cells(FIG. 1A) and KS cells (FIG. 1B) with different doses of microvesicles(MVs).

FIGS. 2A and B graphically show the incubation results of HepG2 and KScells with MVs.

FIGS. 3A and 3B graphically show the incubation results of MCF-7 cells(FIG. 3A) and SKOV-3 cells (FIG. 3B) with 30 μg/ml of MVs from BM-MSCsfor 48 hours.

FIGS. 4A-4C graphically show the MVs from BM-MSCs inducing an increaseof cells in the G0/G1 phase, especially in SKOV-3 cells.

FIGS. 5A and 5B graphically show the results of experiments carried outto evaluate the effect of MVs on in vitro angiogenesis.

FIG. 6 graphically shows the results of experiments carried out toevaluate the effect of MVs on in vitro apoptosis.

FIG. 7 graphically shows the results of experiments carried out toevaluate the effect of MVs on the proliferation of TECs.

FIG. 8 graphically shows the in vivo anti-tumor activity of MVs, treatedor not with RNase, intra-tumor administrated to SCID mice bearing HepG2xenograft tumours.

FIG. 9A shows photographs of mice with HepG2 tumours treated (on theright) or not (on the left) with MVs from BM-MSCs.

FIG. 9B shows photographs of excised HepG2 tumours treated (on theright) or not (on the left) with MVs from BM-MSCs.

FIG. 9C shows microscopic photographs of haematoxylin and eosin stainingof cells of excised HepG2 tumours treated (on the right) or not (on theleft) with MVs from BM-MSCs.

FIG. 10A shows a photograph of excised HepG2 tumours treated withMV-RNase.

FIG. 10B shows a microscopic photograph of haematoxylin and eosinstaining of cells of excised HepG2 tumours treated with MV-RNase.

FIG. 11 is a graph showing the results of the BrdU-based proliferationassay on HepG2.

FIG. 12 is a graph showing the results of apoptosis assays on HepG2cells.

FIG. 13 is a graph showing the results of apoptosis assays on HepG2cells under basal conditions and variously treated HepG2 cells.

FIG. 14 is a graph showing the data obtained by measuring the tumourvolume of recovered HepG2 tumors after various treatments.

FIGS. 15A, 15B, and 15C are micrographs showing the in vivo inhibitionof tumour growth by HLSC-MV treatment and induced intra-tumourapoptosis.

FIG. 16 is a graph showing the results of in vitro proliferation assayscarried out by incubating MCF-7 cells with different concentrations ofHLSC-MVs.

FIG. 17 is a graph showing the results of in vitro proliferation assayscarried out by incubating Kaposi cells (KS) with differentconcentrations HLSC-MVs.

FIG. 18 is a graph showing the results of in vitro apoptosis assayscarried out by incubating MCF-7 cells and Kaposi cells with HLSC-MVs.

The observed anti-tumour activities of microvesicles derived from adultstem cells are quite unexpected over the prior art. Preparations ofmesenchymal stem cells (MSCs) are in fact known to exert a regenerativeeffect on some tissues. For example, bone marrow-derived MSCs are knownto naturally support hematopoiesis by secreting a number of trophicmolecules, including soluble extracellular matrix glycoproteins,cytokines and growth factors. Moreover, microvesicles derived fromendothelial stem cells were shown in WO2009/050742 to promoteangiogenesis and resistance to apoptosis, both in vitro and in vivo. InWO2009/057165 microvesicles derived from stem cells were shown to induceendothelial and epithelial regeneration of damaged tissues or organs.

In WO2009/105044 microvesicles derived from a mesenchymal stem cell arespeculatively said to be suitable for use in the treatment of a highnumber and a great variety of diseases No experimental evidence, notheoretical explanation and no specific method for treatment of canceris provided.

Thus, a first aspect of the present invention is a microvescicle derivedfrom an adult stem cell for use in the therapeutic treatment of a tumourdisease.

In this connection, it should be understood that the microvesicles isnot necessarily derived from adult stem cells taken from the samepatient to which they shall be administered. Rather, they may be derivedfrom a different subject, prepared and maintained in the form of amedicament and then administered to a patient in need thereof. This isthe so-called allogenic approach.

In a preferred embodiment, the adult stem cell is a human mesenchymalstem cell or a human liver stem cell. A preferred human liver stem cellis the human non-oval liver stem cell (HLSC) expressing both mesenchymaland embryonic stem cell markers disclosed in WO 2006/126219. This cellline is in particular characterised in that it is a non-oval human liverpluripotent progenitor cell line isolated from adult tissue whichexpresses hepatic cell markers and which is capable of differentiatinginto mature liver cells, insulin producing cells, osteogenic cells andepithelial cells and preferably express markers selected from the groupcomprising albumin, α-fetoprotein, CK18, CD44, CD29, CD73, CD146, CD105,CD90 and preferably do not express markers selected from the groupcomprising CD133, CD117, CK19, CD34, cytochrome P450.

In another preferred embodiment, the human mesenchymal stem cell isderived from human adult bone marrow (BM-MSC). In another preferredembodiment, the human mesenchymal stem cell is derived from human adultdecapsulated glomeruli (Gl-MSC), as disclosed in European patentapplication no. 08425708.8. These cells are furthermore characterised inthat they are CD133 negative, CD146 positive and CD34 negative and thatthey are capable of differentiating into podocytes, endothelial cellsand mesangial cells and they also preferably express markers selectedfrom the group comprising CD24, Pax-2, CD31, CD29, CD44, CD73, CD90, CD105, CD166, nanog, musashi, vimentin, nestin and preferably do notexpress markers selected from the group comprising α-SMA, Oct-4, CD45,cytokeratin, CD80, CD86, CD40.

According to one embodiment of the invention, the tumour disease isselected from the group consisting of liver tumour (e.g. hepatoma),epithelial tumour (e.g. Kaposi's sarcoma), breast tumour (e.g. breastadenocarcinoma), lung tumour, prostate tumour, gastric tumour, colontumour and ovarian tumour.

Another aspect of the present invention is the use of a microvesciclederived from an adult stem cell as defined above, for preparing amedicament for the therapeutic treatment of a tumour disease as definedabove.

In one embodiment, the therapeutic treatment comprises theadministration of one or more cytotoxic or cytostatic agents. Suitablecytotoxic and cytostatic agents include for example Paclitaxel,Lenalidomide, Pomalidomide, Epirubicin, 5FU, Sunitinib, La-patinib,Canertinib, cyclophosphamide, doxorubicin, Lenalidomiden/Dexamethason,Po-malidomide/Dexamethasone, Carboplatin, Rapamycin, mitoxantron,oxaliplatin, docetaxel, vinorelbin, vincristine and any combinationthereof. The administration of a combination of doxorubicin and/orvincristine with MVs derived from an adult stem cell is highlypreferred, since such a combination has been shown to exert asynergistic effect (see FIG. 13).

The microvescicle is administered to a patient in need thereof eitherlocally or systemically. A pharmaceutical dosage form suitable for bothlocal and systemic administration is e.g. an injectable dosage form. Byway of example, the microvescicle is administered by local intra-tumour(i.t.) injection in a solid tumour, or by i.v. injection or infusion,both in the ease of a solid tumour and in the case of metastasis. Asuitable MV dose to be administered depends on a plurality of factors,but it is generally comprised between 0.1 to 200 micrograms/kg bodyweight of the recipient, preferably 1-150 micrograms/kg body weight ofthe recipient, even more preferably 3-120 micrograms/kg body weight ofthe recipient.

The expression “microvescicle (MV) derived from an adult stem cell” asuse herein refers to a membrane particle which is at least in partderived from an adult stem cell. In turn, the term “adult stem cell”includes any undifferentiated or partially undifferentiated cell whichis capable of proliferating (self-renewal) and differentiating(plasticity), thereby replacing the mature cells of a specialized celllineages which have reached the end of their life cycle.

The term “adult stem cell” as used in the present description includesboth stem cells having unlimited self-renewal ability and pluripotentplasticity, and progenitor cells with multipotent plasticity and, insome instances, a limited self-renewal ability. In a preferredembodiment the “adult stem cells” have pluripotent or multipotentplasticity meaning that they are capable of differentiating into atleast two, more preferably at least three, distinct types ofspecialised, fully differentiated, mature cells.

Within the context of the present description, the expression “adultstem cell” is intended to mean a stem cell that is isolated from anadult tissue, in contrast with an “embryonic stem cell” which isisolated from the inner cell mass of a blastocyst. Adult stem cells arealso known as “somatic stem cells”.

Within the context of the present description, the expression“microvesicles (MV) derived from an adult stem cell is intended to meanthat the microvesicles are directly derived from undifferentiated stemcells.

Within the context of the present description, the expression “directly”means that the microvesicles are derived from the undifferentiated adultstem cells without any differentiation step being carried out or havingtaken place before obtaining of the microvesicles.

The microvesicles derived from adult stem cells used in the presentinvention are generally spheroid in shape and have a diameter within therange of 100 nm to 5 μm, more typically of between 0.2 and 1 μm. If theparticle is not spheroid in shape, the above mentioned values arereferred to the largest dimension of the particle.

The stem cells from which the microvesicles used in the invention areobtained may be isolated as described in the experimental section of thedescription. The microvesicles (MVs) may then be obtained from thesupernatants of the isolated stem cells, e.g. by ultracentrifugation asdisclosed in the experimental section of the description. The isolatedMVs may then be stored until use by freezing at very low temperature,e.g. at −80° C., in a suspension with one or more cryoprotecting agent.Suitable cryoprotecting agents are e.g. dimethylsulphoxide (DMSO) andglycerol. The use of DMSO at a concentration of 1% of the volume of thecell suspension guarantees good preservation of the cells and a limitedtoxic effect on re-infused patients. Other substances which may bementioned as cryoprotecting agents are extracellular cryoprotectingagents, that is to say high molecular weight substances acting at thecell surface forming a tight barrier which reduced intracellulardehydration.

Further objects and advantages of the invention will appear more clearlyfrom the following examples, which are provided by way of illustrationonly. In the example, reference is made to the following figures:

FIGS. 1A and B show that incubation of HepG2 cells (FIG. 1A) and KScells (FIG. 1B) with different doses of microvesicles (MVs)significantly inhibits proliferation compared to control cells incubatedwith vehicle alone. Proliferation of HepG2 and KS cells was evaluated byBrdU incorporation assay after 48 hours of incubation with differentdoses of MVs (1, 10 and 30 μg/ml) from BM-MSCs or from Gl-MSCspre-treated or not with RNase. The results are expressed as the mean±SDof 3 experiments.

FIGS. 2A and B show that incubation of HepG2 and KS cells with MVssignificantly promotes apoptosis compared to control incubated withvehicle alone and in the same way of doxorubicin stimulation. Apoptosisof HepG2 and KS cells was evaluated by Tunel assay as the percentage ofapoptotic cells after 24-hours and/or 48 hours of incubation withdifferent doses of MVs from BM-MSC (pre-treated or not with RNase) orGl-MSC. The results are expressed as the mean±SD of 3 experiments.

FIGS. 3A and 3B show that incubation of MCF-7 cells (FIG. 3A) and SKOV-3cells (FIG. 3B) with 30 μg/ml of MVs from BM-MSCs for 48 hourssignificantly inhibits proliferation compared to control cells incubatedwith vehicle alone. Proliferation of MCF-7 and SKOV-3 cells wasevaluated by BrdU incorporation assay after 48 hours of incubation with30 μg/ml of MVs from BM-MSCs or from Gl-MSCs. The results are expressedas the mean±SD of 3 experiments.

FIG. 4 shows that MVs from BM-MSCs induce an increase of cells in theG0/G1 phase, especially in SKOV-3 cells. The DNA content was measured inSKOV-3 cells cultured with 10% FCS, deprived of PCS (starvation) and inthe presence of 30 μg/ml of MVs for 24 hours. An increase of the numberof cells in the G0/G1 phase in the presence of MVs is observed.

FIGS. 5A and 5B show the results of experiments carried out to evaluatethe effect of MVs on in vitro angiogenesis. The ability of HUVECs andTECs to form capillary-like structures within Matrigel was evaluated.

FIG. 6 shows the results of experiments carried out to evaluate theeffect of MVs on in vitro apoptosis. Apoptosis of HUVECs and TECs wasevaluated by Tunel assay as a percentage of apoptotic cells after 48hours of incubation with different doses of MVs.

FIG. 7 shows the results of experiments carried out to evaluate theeffect of MVs on the proliferation of TECs. The proliferation of TECswas evaluated by BrdU incorporation assay after 48 hours of incubationwith different doses of MV (10 and 30 μg/ml) from BM-MSC pre-treated ornot with RNase. The results are expressed as the mean±SD of 2experiments.

FIG. 8 shows the in vivo anti-tumor activity of MVs, treated or not withRNase, intra-tumor administrated to SCID mice bearing HepG2 xenografttumours. Tumor mass was determined by caliper measurement of twoperpendicular diameters of the implant every week. The results are shownas the percentage of increment of tumour mass: the tumour mass at thefirst treatment (1 week after HepG2 injection) is fixed by convention asthe 100% value.

FIG. 9 shows that MVs derived from BM-MSCs reduce tumor growth in vivo.A) Representative examples of mice with HepG2 tumours treated (on theright) or not (on the left) with MVs from BM-MSCs. B) Representativeexamples of excised HepG2 tumours treated (on the right) or not (on theleft) with MVs from BM-MSCs. C) Haematoxylin and Eosin staining of HepG2tumours treated (on the right) or not (on the left) with MVs fromBM-MSCs.

FIG. 10 shows that pre-treatment of MVs with RNase abrogates theanti-tumor activity of MVs derived from BM-MSCs. A) Representativeexamples of excised HepG2 tumours treated with MV-RNase. B) Haematoxylinand Eosin staining of HepG2 tumours treated with MV-RNase.

FIG. 11 is a graph showing the results of the BrdU-based proliferationassay on HepG2. HepG2 were cultured in DMEM alone or supplemented withdifferent doses of MVs derived from HLSCs. After 3 days, HepG2proliferation was quantified using the BrdU incorporation assay.

FIG. 12 is a graph showing the results of apoptosis assays on HepG2cells. HepG2 cells were cultured in DMEM only or with 10% FCS andsupplemented with vincristine (100 ng/ml), or MV-HLSC (30 μg/ml), orMV-HLSC (pre-treated with RNase, 30 μg/ml). The analysis was performedafter 24 hours.

FIG. 13 is a graph showing the results of apoptosis assays on HepG2cells under basal conditions or HepG2 cells treated with vincristine,doxorubicin, MV-HLSCs, or with vincristine plus MV-HLSCs and doxorubicinplus MV-HLSCs.

FIG. 14 is a graph showing the data obtained by measuring the tumourvolume of recovered HepG2 tumors after MV-HLSC (n=3), vehicle (n=2) orMV-HLSC (n=3) RNAse treated, i.t. treatment at time of mice sacrifice.Tumour volume was determined by measuring with a caliper twoperpendicular diameters of the implant every week.

FIG. 15 shows micrographs showing the in vivo inhibition of the tumourgrowth by HLSC-MV treatment and the induced intra-tumour apoptosis. A)Representative micrographs showing apoptosis, PCNA and Haematoxylin &Eosin staining of recovered HepG2 tumours after 4 weeks. B)Representative micrographs showing apoptosis, PCNA and Haematoxylin &Eosin staining of recovered HepG2 tumors from MV-treated mice. C)Representative micrographs showing apoptosis, PCNA and Haematoxylin &Eosin staining of recovered HepG2 tumors from MV-RNAse treated mice.

FIG. 16 is a graph showing the results of in vitro proliferation assayscarried out by incubating MCF-7 cells with different concentrations ofHLSC-MVs. Proliferation of MCF-7 cells was evaluated by BrdUincorporation assay after 48 hours of incubation with 2, 10, 15 and 30μg/ml of MVs derived from HLSC cells. The experiment was performed intriplicate. P< 0.05.

FIG. 17 is a graph showing the results of in vitro proliferation assayscarried out by incubating Kaposi cells (KS) with differentconcentrations HLSC-MVs. Proliferation of Kaposi cells was evaluated byBrdU incorporation assay after 48 hours of incubation with 2, 10, 15 and30 μg/ml of MVs derived from HLSC cells. The experiment was performed intriplicate. P<0.05.

FIG. 18 is a graph showing the results of in vitro apoptosis assayscarried out by incubating MCF-7 cells and Kaposi cells with HLSC-MVs.Apoptosis was evaluated by TUNEL assay as the percentage of apoptoticcells after 48-hours of incubation with different doses of MVs (2; 10;15: and 30 μg/ml and 30 μg/ml of RNase treated MVs). Doxorubicin wereused as the positive control of apoptosis induction. In the negativecontrol, MCF-7 cells and Kaposi cells were treated with vehicle alone.The experiment was performed in triplicate. P<0.05.

1. Microvesicles (MVs) from Mesenchymal Stem Cells (MSCs)

1.1 Isolation and Characterization of MSCs

Bone marrow cells were layered on a Ficoll gradient (density: 1022 g/ml;Sigma-Aldrich, St. Louis, Mo.) and centrifuged at 1500 rpm for 30minutes. The mononucleated cells were cultured in the presence ofMesenchymal Stem Cells Basal Medium (MSCBM, Lonza). After 5 days ofculture, the medium was changed. To expand the isolated cells, theadherent monolayer was detached by trypsin treatment for 5 minutes at37° C., on day 15 for the first passage and every 7 days for thesubsequent passages. The cells were seeded at a density of 10,000cells/cm² and used not later than passage 6.

MSC populations from glomeruli (Gl-MSC) were obtained from the normalportion of cortex from surgically removed kidneys, as described in BrunoS et al. Isolation and characterization of resident mesenchymal stemcells in human glomeruli. Stem Cells Dev. 2009; 18:867-880. Afterdissection of the cortex, the glomerular suspension was collected usinga standard established technique: after passing through a graded seriesof meshes (60 and 120 meshes), glomeruli were recovered at the top ofthe 120 meshes sieve. Glomeruli were then collected at the bottom of aconical tube by spontaneous precipitation (10 minutes at roomtemperature) and were deprived of the visceral layer of the Bowman'scapsule both mechanically, by several rounds of aspiration/expulsionusing a 10 ml pipette, and enzymatically, by digestion for 2 minuteswith Collagenase I (Sigma, St. Louis, Mo.). Glomeruli were thencollected at the bottom of a conical tube by spontaneous precipitationin order to remove cells and Bowman capsules, and were transferred tofibronectin coated T25 flasks (Falcon, BD Bioscience, Two Oak Park,Bedford, Mass.). Glomeruli were cultured in the presence of MesenchymalStem Cells Basal Medium (MSCBM, Lonza). Cells were left to reachconfluence prior to passaging; the interval between passages varied (3-7days) until passage 4, and from then on it was established at around 7days.

At each passage, the cells were counted and analyzed for immunophenotypeby cytofluorimetric analysis and immunofluorescence. Cytofluorimetricanalysis was performed with the following antibodies, all phycoerythrin(PE) or fluorescein isothiocyanate (FITC) conjugated: anti-CD105, -CD29,-CD31, -CD146, -CD44, -CD90 (Dakocytomation, Copenhagen, Denmark);-CD73, -CD34, -CD45, -CD80, -CD86, -CD166, HLA-I (Becton DickinsonBiosciences Pharmingen, San Jose, Calif.); -CD133 (Miltenyi Biotec,Auburn); KDR (R&D Systems, Abington, U.K.); -HLA-II (ChemiconInternational Temecula, Calif.), -CD40 (Immunotech, Beckman Coulter),-CD 154 (Serotec, Raleigh, N.C. USA) monoclonal antibodies. Mouse IgGisotypic controls were from Dakocytomation. All incubations wereperformed in 100 μl of phosphate buffered saline (PBS) containing 0.1%bovine serum albumin and 0.1% sodium azide, at 4° C. For each sample,10,000 cells were analysed on FACSCalibur cytometer (BD BiosciencesPharmingen). Gating was constructed based on negative controls andcompensation controls were included in all analyses performed.Population percentages and numbers were generated for gated populationsfrom each experiment using Cell Quest software (BD BiosciencesPharmingen).

Indirect immunofluorescence was performed on MSCs cultured on chamberslides (Nalgen Nunc International, Rochester, N.Y., USA), fixed in 4%paraformaldehyde containing 2% sucrose and, if required, permeabilizedwith Hepes-Triton X 100 buffer (Sigma, St. Louis, Mo.). The followingantibodies were used: mouse monoclonal anti-vimentin (Sigma) and rabbitpoliclonal anti-von Willebrand factor (Dakocytomation). Omission of theprimary antibodies or substitution with non immune rabbit or mouse IgGwere used as controls where appropriated. Alexa Fluor 488 anti-rabbitand anti-mouse Texas Red (Molecular Probes, Leiden, The Netherlands)were used as secondary Abs. Confocal microscopy analysis was performedusing a Zeiss LSM 5 Pascal Model Confocal Microscope (Carl ZeissInternational, Germany). Hoechst 33258 dye (Sigma) was added for nuclearstaining.

BM-MSC and GL-MSC preparations did not express hematopoietic markerssuch as CD45, CD14 and CD34. They neither expressed the co-stimulatorymolecules (CD80, CD86 and CD40) and the endothelial markers (CD31, vonWillebrand Factor, KDR). All the cell preparations at different passagesof culture expressed the typical MSC markers: CD105, CD73, CD44, CD90,CD166 and CD146. They also expressed HLA class I.

The adipogenic, osteogenic and chondrogenic differentiation abilities ofMSC were determined as described in Pittenger M F, Martin B J.Mesenchymal stem cells and their potential as cardiac therapeutics.Circ. Res 2004;95:9-20. In brief, MSCs were cultured with AdipogenicMedium (Lonza) for 3 weeks. To evaluate the differentiation, the cellswere fixed with 4% paraformaldehyde for 20 minutes at room temperatureand stained with 0.5% Oil Red O (Sigma) in methanol (Sigma) for 20minutes at room temperature.

Osteogenic differentiation was assessed by culturing MSCs in OsteogenicMedium (Lonza). The medium was changed twice per week for 3 weeks. Toevaluate the differentiation, cells were fixed with 4% paraformaldehydefor 20 minutes and stained with Alizarin Red, pH 4.1 (Lonza) for 20minutes at room temperature.

For chondrogenic differentiation, 2.5×10⁵ MSCs were centrifuged in a15-ml conical polypropylene tube (Falcon BD Bioscience) at 150 g for 5minutes and washed twice with DMEM. The pellets were cultured inChondrogenic medium (Lonza) supplemented with 10 ng/ml of TransformingGrowth Factor β3 (Lonza). The medium was changed every 3 days for 28days. Pellets were fixed in 4% paraformaldehyde overnight, and theparaffin-embedded sections were stained for glycosaminoglycans using0.1% safranin O (Sigma) and for sulfated proteoglycans using 1% alcianblue.

1.2 Isolation and Characterization of MVs Derived from MSCs

The microvesicles (MVs) were obtained from supernatants of BM-MSCs orGL-MSCs obtained as describe above cultured in RPMI deprived of PCS andsupplemented with 0.5% of BSA (Sigma). After centrifugation at 2,000 gfor 20 minutes to remove debris, cell-free supernatants were centrifugedat 100,000 g (Beckman Coulter Optima L-90K ultracentrifuge) for 1 hourat 4° C., washed in serum-free medium 199 containingN-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES) 25 mM(Sigma) and subjected to a second ultracentrifugation under the sameconditions. Endotoxin contamination of MVs was excluded by Limulus testaccording to the manufacturer's instructions (Charles RiverLaboratories, Inc., Wilmington, Mass., USA) and MVs were stored at −80°C.

In selected experiments, MVs were treated with 1 U/ml RNase (AmbionInc., Austin, Tex., USA) for 1 hour at 37° C. the reaction was stoppedby addition of 10 U/ml RNase inhibitor (Ambion Inc.) and MVs were washedby ultracentrifugation.

1.3 In Vitro Experiments Performed with MVs Derived from MSCs

Cancer cell lines culture. Human liver cancer cell line (HepG2), humanbreast adenocarcinoma cell line (MCF-7) and human ovarian cancer cellline (SKOV-3) were cultured in low glucose DMEM (Euroclone) containing10% of Fetal Calf Serum (FCS, Euroclone), 100 U/ml penicillin, 100 mg/mlstreptomycin and 1% glutamine (all from Sigma) and maintained in anincubator with a humidified atmosphere of 5% CO₂ at 37° C. A primaryculture of Kaposi's sarcoma cells (KS cells) was obtained from acutaneous lesion of a patient bearing renal allograft underimmunosuppressive therapy and cultured in RPMI 1640 medium supplementedwith 10% of FCS, 100 μg/ml penicillin, and 100 μg/ml streptomycin.

Tumor endothelial cells (TECs): isolation and culture. TECs wereisolated from specimens of clear-cell type renal cell carcinomas usinganti-CD 105 Ab coupled to magnetic beads by magnetic cell sorting usingthe MACS system (Miltenyi Biotec, Auburn, Calif., USA). TEC cell lineswere established and maintained in culture in endothelial basal completemedium (EBM) supplemented with epidermal growth factor (10 ng/ml),hydrocortisone (1 mg/ml), bovine brain extract (all from Lonza) and 10%FCS. TECs were characterized as endothelial cells by morphology,positive staining for vWF antigen, CD 105, CD146, and vascularendothelial-cadherin and negative staining for cytokeratin and desmin(Bussolati B et al. Altered angiogenesis and survival in endothelialcells derived from renal carcinoma. FASEB J 2003; 17:1159-1161).

Humbelical Vein Endothelial Cells (HUVECs): isolation and culture. Humanumbilical vein endothelial cells (HUVECs) were obtained from theumbilical vein, as described previously (Bussolati B et al. Vascularendothelial growth factor receptor-1 modulates vascular endothelialgrowth factor-mediated angiogenesis via nitric oxide. Am J Pathol. 2001September; 159(3):993-1008) and maintained in EBM and 10% FCS.Experiments were performed on second or third passage HUVECs.

Cell Proliferation. HepG2, MCF-7, SKOV-3, KS cells or HUVECs were seededat 2,000 or 4,000 cells/well into 96-well plates in DMEM (Sigma)deprived of FCS with different concentrations of microvesiclespre-treated or not pre-treated with RNase. DNA synthesis was detected asincorporation of 5-bromo-2′-deoxy-uridine (BrdU) into the cellular DNAafter 48 hours of culture. Cells were then fixed with 0.5 M ethanol/HCland incubated with nuclease to digest the DNA. BrdU incorporation intothe DNA was detected using an anti-BrdU peroxidase-conjugated mAb andvisualized with a soluble chromogenic substrate (Roche Applied Science,Mannheim, Germany). Optical density was measured with an ELISA reader at405 nm.

Cell cycle analysis. Human cancer cell lines were stimulated for 24hours with 30 μg/ml of different preparations of MVs, detached bytrypsin and fixed in cold 80% ethanol. Cells were maintained for atleast 24 hours at −20° C. and then washed in PBS. Then, the cells wereincubated for 1 hour at room temperature with propidium iodide (50μg/ml) (Sigma) to stain the DNA in a solution containing RNase (200μg/ml) (Sigma) and 0.5% of Nonidet P40 (Sigma). For each sample, 50,000cells were analysed on FACSCalibur cytometer (BD BiosciencesPharmingen).

Apoptotic assay. HepG2, MCF-7, SKOV-3, KS cells, HUVECs or TECs wereseeded at 8,000 cells/well into 96-well plated in DMEM (Sigma) with 10%FCS and in the presence of doxorubicin (100 ng/ml, Sigma) or differentconcentrations of MVs (10 and 30 μg/ml) pre-treated or not with RNase.Apoptosis was assessed by TUNEL assay (ApopTag Oncor, Gaithersburg, Md.,USA). After 24 or 48 hours of treatment, the cells were washed with PBS,fixed in 1% paraformaldehyde pH 7.4 for 15 minutes at 4° C., washedtwice in PBS and then post-fixed in pre-cooled ethanol-acetic acid 2:1for 5 minutes at −20° C. The samples were treated with the enzymeterminal deoxynucleotidyl transferase (TdT). The cells were then treatedwith heated anti-digoxigenin conjugate with fluorescein and incubatedfor 30 minutes at room temperature. The samples were mounted in mediumcontaining 1 μg/ml of propidium iodide and the cells analyzed byimmunofluorescence. The results are expressed as the percentage of greenfluorescence emitting cells (apoptotic cells) versus red fluorescenceemitting cells (total cells).

Angiogenesis in vitro. 24-well plates were coated with growthfactor-reduced Matrigel (BD Biosciences) at 4° C. and incubated for 30minutes at 37° C., 5% CO₂, in a humidified atmosphere. HUVECs or TECswere seeded on the Matrigel-coated wells in RPMI or EBM with 5% FCS atthe density of 5×10⁴ cells/well, in the presence or in the absence ofdifferent concentrations of MVs treated or not treated with RNase. After6 hours of incubation, the cells were observed under a Nikon invertedmicroscope (Nikon) and the experimental results were recorded. Theresults were expressed as the mean of the tube length, measured with theMicroimage analysis system (Cast Imaging), expressed in arbitrary unitsand evaluated in 5 different fields at a magnification of 20× induplicate wells from 3 different experiments.

Statistical analysis. All data from different experimental proceduresare expressed as the mean±SD. Statistical analysis was performed byANOVA with Newmann-Keuls multi-comparison test where appropriate.

1.4 In Vitro Results

1.4.1 In Vitro Biological Effects of MVs Derived from BM-MSCs andGL-MSCs on Tumour Cell Lines

The anti-tumour activity of MVs derived from human BM-MSCs, was assessedin vitro by measuring their ability to inhibit proliferation and toinduce apoptosis on HepG2, MCF-7, SKOV-3 and KS cell lines.

FIG. 1 shows that incubation of HepG2 cells (FIG. 1A) and KS cells (FIG.1B) with different doses of MVs for 48 hours significantly inhibitedproliferation compared to control cells incubated with vehicle alone.

FIG. 2 shows that incubation of HepG2 cells (FIG. 2A) and KS cells (FIG.2B) with MVs for 24 and 48 hours significantly promoted apoptosiscompared to control cells incubated with vehicle alone and in the sameway of doxorubicin stimulation.

When MVs were incubated with RNase to induce complete degradation of theRNA shuttled by MVs, the anti-proliferation and pro-apoptotic effectselicited by MVs on HepG2 and KS cells were reduced (FIGS. 1 and 2).RNase treatment of MVs did not interfere per se with cancer cell lineapoptosis induced by doxorubicin (FIG. 2).

On the contrary, incubation of MCF-7 cells and SKOV-3 cells with 30μg/ml of MVs from BM-MSCs for 48 hours significantly inhibitedproliferation compared to control cells incubated with vehicle alone(FIGS. 3A and 3B), but did not promote apoptosis. In these two tumourcell lines the inventors have also studied cell cycle with the propidiumiodide staining technique, in order to evaluate the percentage of cellsin the G0/G1 phase in comparison with the cells in the S and G2 phases.The inventors have observed that MVs from BM-MSCs induced an increase ofcells in the G0/G1 phase, especially in SKOV-3 cells (FIG. 4), which mayexplain the inhibition of proliferation observed with BrdUincorporation.

The inventors have also tested the effects of MVs derived from Gl-MSCson the proliferation and apoptosis of tumor cell lines. Gl-MSCs did notaffect proliferation and apoptosis of the HepG2 cell line. On thecontrary, MVs derived from Gl-MSCs inhibited proliferation and inducedapoptosis of KS cells (FIGS. 1 and 2). Moreover, incubation of MCF-7 andSKOV-3 cells with 30 μg/ml of MVs derived from Gl-MSCs for 48 hoursinhibited proliferation compared to control cells incubated with vehiclealone (FIG. 3), but did not promote apoptosis.

1.4.2 MVs Derived from Human Fibroblasts

MVs derived from human fibroblasts did not inhibit proliferation and didnot induce apoptosis of different cancer cell lines (data not shown).

1.4.3 In Vitro Effects of MVs Derived from BM-MSCs on Endothelial Cells

The inventors have also studied the in vitro effects of MVs derived fromBM-MSCs on the proliferation, apoptosis and capillary-like formation ofHUVECs and tumour endothelial cells (TECs).

MV-treatment did not affect the proliferation (data not shown) and thecapillary-like formation ability of HUVECs (FIG. 5A). in addition,incubation of HUVECs with different doses of MVs for 48 hours did notinduce apoptosis (FIG. 6).

In contrast, the incubation of TECs with different doses of MVs for 48hours, significantly inhibited proliferation (FIG. 7) and promotedapoptosis (FIG. 6) compared to the control cells incubated with vehiclealone. Proliferation of TECs was evaluated by BrdU incorporation assayafter 48 hours of incubation with different doses of MVs (10 and 30μg/ml) from BM-MSCs pre-treated or not with RNase. The results in FIG. 7are expressed as the mean±SD of 2 experiments.

When MVs were incubated with RNase, the apoptotic effect elicited by MVson TECs was significantly reduced. Incubation of TECs seeded on Matrigelwith different doses of MVs significantly inhibited the ability of TECsto form capillary-like structures in vitro. Pre-treatment of MVs withRNase abrogated the inhibitory effect on MVs on tubule formation (FIG.5B).

1.5 In Vivo Experiments with MVs Derived from MSCs

Tumor formation, 3×10⁶ HepG2 cells were collected and implantedsubcutaneously into SCID mice (Charles River, Jackson Laboratories, BarHarbor, Me.). Cultured cells, harvested using trypsin-EDTA, were washedwith PBS, counted in a microcytometer chamber and resuspended in 100 μlof DMEM and 100 μl of Matrigel Matrix (Becton Dickinson). The cells werechilled on ice, and injected subcutaneously into the left back of SCIDmice via a 26-gauge needle using a 1-ml syringe. The animals weremonitored for activity and physical conditions every day, and thedetermination of body weight and measurement of tumour mass was madeevery 3 days. Tumour mass was determined by caliper measurement in twoperpendicular diameters of the implant and calculated using the formula½a×b², where a is the long diameter and b is the short diameter (Hou Jet al. Experimental therapy of hepatoma with artemisin and itsderivatives: in vitro and in vivo activity, chemosensitization andmechanism of action. Clin Cancer Research. 2008; 14:5519-5530)). After 1week, when the implanted tumours reached the volume of approximately 15mm³, the inventors started the weekly intra-tumour injection of MVs. Thefirst treatment was with 100 μg of MVs (treated or not with RNase) for amaximum volume of 20 μl the subsequent intra-tumour injections were of50 μg of MVs (treated or not with RNase), for a maximum of 20 μl. Incontrol mice, the inventors injected intra-tumour the same volume ofvehicle alone. Mice were randomized into three treatment groups: a) thegroup intra-tumor injected with of MVs (n=8); b) the group intra-tumorinjected with MVs treated with RNase (n=8); and c) the control groupinjected with the same volume of vehicle alone (n=5). After three weeksfrom Matrigel injection, mice were sacrificed and tumours recovered andprocessed for histology.

1.6 In Vivo Results

1.6.1 In Vivo Biological Effects of MVs Derived from BM-MSCs on HEPG2Tumor Growth

Tumor formation and growth are inhibited by MVs derived form BM-MSCs inSCID mice. To determine the effect of MVs derived from BM-MSCs on tumorformation and growth in vivo, SCID mice were injected subcutaneouslywith HepG2 in the presence of Matrigel. One week after the injection,when the volume of tumours was about 15 mm³, the inventors started toweekly inject the mice intra-tumour with MVs (treated or not withRNase), with a maximum volume of 20 μl. The first treatment was with 100μg of MVs; the subsequent intra-tumour injections were with 50 μg ofMVs. In control mice, the inventors injected inject 20 μl of vehiclealone intra-tumour.

After three weeks from Matrigel injection, all tumours were recoveredand analyzed. In the HepG2 xenograft model, MV intra-tumor injectionshowed a inhibitor effect on tumor growth (FIG. 8). Tumor size andvolume were significantly smaller in SCID mice treated with MVs (FIGS.9A and B) and histological analyses showed areas of necrosis in HepG2tumours treated with MVs (FIG. 9C). Tumours injected with MVspre-treated with RNase did not differ in size and histology from controltumours (FIGS. 10A and B).

2. Microvesicles (MVs) from Liver Stem Cells

2.1 Isolation and Characterization of Adult Human Liver Stem Cells(HLSCs)

HLSCs were isolated from human cryopreserved normal hepatocytes obtainedfrom Cambrex Bio Science Verviers S.p.r.l. (Verviers, Belgium) culturedin minimum essential medium/endothelial cell basal medium-1 (α-MEM/EBM)(3:1) (Gibco/Cambrex) supplemented with L-glutamine (5 mM), Hepes (12mM, pH 7.4), penicillin (50 IU/ml), streptomycin (50 μg/ml) (all fromSigma, St. Louis), FCS (10%). The expanded cells were transferred to aT-75 flask and analyzed when they approached confluence.

The hepatoma cell line HepG2 were cultured in Dulbecco's modifiedEagle's medium (DMEM) (low glucose) containing 10% fetal bovine serum(FBS).

2.2 Isolation of MVs Derived from HLSCs

MVs were obtained from supernatants of HLSCs cultured in MEM-alphasupplemented with 2% of Fetal Bovine Serum (FBS). The viability of cellsincubated overnight without serum was detected by trypan blue exclusion.After centrifugation at 2000 g for 20 minutes to remove debris,cell-free supernatants were centrifuged at 100,000 g (Beckman CoulterOptima L-90K ultracentrifuge) for 1 h at 4° C., washed in serum-freemedium 199 containing N-2-hydroxyethylpiperazine-N′-2-ethanesulfonicacid (HEPES) 25 mM (Sigma) and subjected to a second ultracentrifugationunder the same conditions. To trace, in vitro and in vivo, MVs byfluorescence microscopy or FACS analysis, MVs from stem cells werelabelled with the red fluorescence aliphatic chromophore intercalatinginto lipid bilayers PKH26 dye (Sigma). After labelling, MVs were washedand ultracentrifuged at 100,000 g for 1 h at 4° C., MV pellets weresuspended in medium 199, and the protein content was quantified by theBradford method (BioRad, Hercules, Calif.). Endotoxin contamination ofMVs was excluded by Limulus testing according to the manufacturersinstructions (Charles River Laboratories. Inc., Wilmington, Mass.), andMVs were stored at −80° C. The morphologic analysis performed on MVsuspension after staining with propidium iodide did not show thepresence of apoptotic bodies.

In selected experiments, MVs from HLSCs were treated with 1 U/ml RNase(Ambion Inc., Austin, Tex.) for 1 h at 37° C., the reaction was stoppedby the addition of 10 U/ml RNase inhibitor (Ambion Inc.) and MVs werewashed by ultracentrifugation. The effectiveness of RNase treatment wasevaluated after RNA extraction using TRIZOL reagent (Invitrogen,Carlsbad, Calif.) by spectrophotometer analysis of total extracted RNA(untreated: 1.3±0.2 μg RNA/mg protein MV; RNase treated: <0.2 μg RNA/mgprotein MV). In addition, RNA extracted from RNase-treated and untreatedMVs was labelled by oligo dT driven retrotranscription and analyzed on0.6% agarose gel to show the complete degradation of RNA by RNasetreatment. As a control, MVs were treated with 1 U/ml DNase (AmbionInc.) for 1 h at 37° C.

2.3 In Vitro Experiments Performed with MVs Isolated from HLSCs

Proliferation assay. DNA synthesis was detected as the incorporation of5-bromo-2-deoxyuridine (BrdU) into the cellular DNA using anenzyme-linked immunosorbent assay kit (Chemicon, Temecula, Calif.)according to the manufacturer's instructions. Briefly, after washing,the cells were incubated with 10 mol/l BrdU for 6 to 12 hours at 37° C.,5% CO₂, in a humidified atmosphere, fixed with 0.5 mol/L ethanol/HCl andincubated with nuclease to digest DNA. BrdU incorporated into the DNAwas detected using an anti-BrdU peroxidase-conjugated monoclonalantibody and visualized with a soluble chromogenic substrate. Opticaldensity was measured with an enzyme-linked immunosorbent assay reader at405 nm.

Apoptosis assay. Apoptosis was evaluated using the terminal dUTP nickendlabeling assay (ApoTag; Oncor, Gaithersburg, Md.). Cells (8×10³/well)were cultured in 96-well plate, suspended in phosphate-buffered saline(PBS) and fixed in 1% paraformaldehyde in PBS, pH 7.4, for 15 minutes at4° C. followed by pre-cooled ethanol/acetic acid (2:1) for 5 minutes at−20° C. Cells were treated with terminal deoxynucleotide transferaseenzyme and incubated in a humidified chamber for 1 hour at 37° C. andthen treated with warmed fluorescein isothiocyanate-conjugatedantidigoxigenin for 30 minutes at room temperature. After washing,samples were mounted in a medium containing 1 g/ml of propidium iodideand the cells were analyzed by immunofluorescence.

Statistical analysis. All data of different experimental procedures areexpressed as average±SD. Statistical analysis was performed by ANOVAwith Newmann-Keuls multi-comparison test where appropriate.

2.4 In Vitro Results

2.4.1 Effects of MVs Derived from HLSCs on the Proliferation of theHepG2 Hepatoma Cell Line

The inventors evaluated the effects of MVs derived from HLSCs on HepG2proliferation. Briefly, HepG2 cells were incubated with different doses(10, 20 and 30μg/ml) of HLSC-derived MVs as such or treated with RNasefor 3 days. At the end of incubation, HepG2 cultures were counted orfixed in 0.5 M ethanol/HCl and incubated with nuclease to digest theDNA. BrdU incorporated into the DNA was detected using an anti-BrdUperoxidase-conjugated mAb and visualized with a soluble chromogenicsubstrate. Optical density was measured with an ELISA reader at 405 nm.As shown in FIG. 11, MVs derived from HLSCs are able to inhibitsignificantly HepG2 proliferation. This also applies to RNase treatedMVs.

2.4.2 Effects of MVs Derived from HLSCs on the Apoptosis of the HepG2Hepatoma Cell Line

The ability of HLSC-derived MVs to induce apoptosis on HepG2 wasevaluated. Briefly, HepG2 were seeded at a density of 8,000 cells/wellinto 96-well plates in DMEM with 10% FCS and apoptosis was induced byculture in the absence of FCS, by treatment with vincristine (100ng/ml), or doxorubicin (50 ng/ml), two mitotic inhibitors used in cancerchemotherapy, or by MV treatment (30 μg/ml). As a control, MVs were alsotreated with 1 U/ml RNase 18 (Ambion, Austin, Tex.) for 1 hour at 37° C.to assess whether the contribution to the inhibition of cancer cellsgrowth is dependent to an horizontal transfer of mKNA delivered by MV tothe cancer cells. Apoptosis was evaluated using the TUNEL assay analysisat 24 and 72 hours. As shown in FIG. 12, MVs derived from HLSCs wereable to induce HepG2 apoptosis comparable to that induced byvincristine. On the contrary, the RNase treatment failed to induceapoptosis. In addition, treatment of HepG2 with vincristine plusMV-HLSCs or doxorubicin plus MV-HLSCs results in an additive effect asshown in FIG. 13.

2.5 In Vivo Experiment Performed with MVs Isolated from HLSCs

Cell Culture. Human hepatoma cells HepG2, were cultured in DMEMsupplemented with 10% fetal bovine serum, 100 μg/ml penicillin, and 100μg/ml streptomycin and maintained in an incubator with a humidifiedatmosphere of 5% CO₂ at 37° C.

Human liver stem cells (HLSCs) were cultured in α-MEM/EBM (3:1),supplemented with 10% fetal bovine serum, 100 μg/ml penicillin, and 100μg/ml streptomycin. EBM was reconstituted with hEGF (human EpithelialGrowth Factor), Hydrocortisone, GA (gentamicin), BBE (Brain BovineExtract).

Isolation of microvesicles (MVs) from HLSCs. MVs were obtained fromsupernatants of HLSCs cultured in α-MEM medium supplemented with 2%fetal bovine serum. After centrifugation at 2,000 g for 20 minutes toremove debris, cell-free supernatants were centrifuged at 100,000 g for1 hour at 4° C., washed in serum-free medium 199 containingN-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES) 25 mM andsubmitted to a second ultracentrifugation under the same conditions. MVpellets were suspended in medium 199 in 0.1% of DMSO and the proteincontent was quantified using the Bradford method.

Experimental design. Male 4- to 5-mo-old SCID mice were obtained fromCharles River laboratories. All mice were housed in a clean facility andheld for 1 week to acclimatize. On day 0, two injections of 3×10⁶ HepG2tumour cells resuspended in serum-free DMEM with Matrigel basementmembrane matrix at a 1:1 ratio were applied. Cell suspension wasinjected in a total volume of 0.2 ml into the left inguinal area of theSCID mice. All mice were randomized into three treatment groups: 20 μlof intratumoral (i.t.) MV injection (n=3); 20 μl of i.t. PBS injection(n=2) and 20 μl of i.t. MV-RNase treated injection (n=3). On day 7post-tumour cell transplantation treatment started. Tumours becamepalpable as from day 7; 50 or 100 μg of MV, suspended in M199supplemented with 0.1%) of DMSO, were injected 7, 12, 14 and 18 daysafter tumour transplantation. Treatment started when tumours rinse thevolume of approximately 15 mm³. The animals were monitored for activityand physical condition everyday, and the determination of body weightand measurement of tumour mass were done every 3 days.

Tumours were measured with callipers. Tumour mass was determined bycalliper, measurement in two perpendicular diameters of the implant andcalculated using the formula ½a×b², where a is the long diameter and bis the short diameter. The animals were sacrificed on day 28, andtumours were collected for further analysis.

Morphological studies. Tumours were fixed in 10% buffered neutralformalin, routinely processed, embedded in paraffin, sectioned at 5 μm,and stained with H&E for microscopic examination. Immunohistochemistryfor detection of proliferation was performed using the anti-PCNAmonoclonal antibody. Sections were blocked and labeled with anti-mouseHRP secondary antibody (1:300 dilution). Omission of the primaryantibodies or substitution with non immune mouse IgG was used ascontrols. Apoptosis was evaluated in paraffin-embedded tumor sections byTUNEL. Ten non consecutive sections were counted for apoptotic-positivetumor cells at 630× magnification. Hoechst 33258 dye was added fornuclear staining.

Statistical analysis. All data of different experimental procedures areexpressed as mean±SD. Statistical analysis was performed by ANOVA withNewmann-Keuis multi-comparison test where appropriate.

2.6 In Vivo Results

2.6.1 Tumour Growth and Proliferation Were Inhibited by MVs Derived fromHLSCs in a Hepatoma Xenograft Model in SCID Mice

To determine the effect of MVs derived from HLSC son tumour growth invivo, SCID mice were subcutaneously transplanted with the humanhepatocarcinoma cell line HepG2. One and two weeks after the injectionof HepG2, when the volume of tumours was about 15 mm³, mice were treatedwith intra-tumour injection of MVs (50 or 100 μg), for a maximum of 20μl of volume. In control mice, tumour were injected with 20 μl ofvehicle alone. After three and four weeks from HepG2 injection, alltumours were recovered and analyzed. In this xenograft model,intra-tumor injection of MVs (FIG. 14) showed a inhibitor effect ontumour growth. In addition, histological analyses showed areas ofnecrosis in tumours treated with MVs (FIG. 15B) and antiproliferativeeffect, was observed using PCNA staining (FIG. 15B).

2.6.2 Induction of Apoptosis by MVs Derived from HLSCs in a HepatomaXenograft Model in SCID Mice

To determine the effect in intra-tumour apoptosis, paraffin sectionsfrom tumours treated with MVs were analysed by TUNEL. MV treatmentinduced apoptosis compared to tumours treated with vehicle alone (FIG.15A).

2.7 In Vitro Biological Effect of MVs derived from HLSCs on MCF-7 BreastAdenocarcinoma and Kaposi's Sarcoma (KS) Cells

2.7.1 Materials and Methods

Cell Culture. Human non oval liver stem cells (HLSC) were cultured inα-MEM/EBM (3:1), supplemented with 10% fetal bovine serum, 100 μg/mlpenicillin and 100 μg/ml streptomycin. EBM was reconstituted with hEGF(human Epithelial Growth Factor), Hydrocortisone, GA (gentamicin), BBE(Brain Bovine Extract).

MCF-7 breast adenocarcinoma cell lines were obtained from American TypeCulture Collection (Manassas, Va.) and were cultured in DMEMsupplemented with 10% of FCS, 100 μg/ml penicillin and 100 μg/mlstreptomycin and maintained in an incubator with a humidified atmosphereof 5% CO₂ at 37° C.

A primary culture of Kaposi's sarcoma cells (KS cells) was obtained froma cutaneous lesion of a patient bearing renal allograft underimmunosuppressive therapy and was cultured in RPMI 1640 mediumsupplemented with 10% of FCS, 100 μg/ml penicillin and 100 μg/mlstreptomycin.

Isolation of MVs. MVs were obtained from supernatants of HLSCs culturedin MEM-alpha supplemented with 2% of Fetal Bovine Serum (FBS) for 18hours. In selected experiments, MVs were collected in the absence ofFBS. The viability of cells incubated overnight at 2% of FBS and withoutserum was detected by trypan blue exclusion (more than 90%, data notshown). After centrifugation at 2,000 g for 20 minutes to remove debris,cell-free supernatants were centrifuged at 100,000 g (Beckman CoulterOptima L-90K ultracentrifuge) for 1 h at 4° C., washed in serum-freemedium 199 containing N-2-hydroxyethylpiperazine-N′-2-ethanesulfonicacid (HEPES) 25 mM and subjected to a second ultracentrifugation underthe same conditions. MV pellets were suspended in medium 199, and theprotein content was measured with the Bradford method. MVs were storedat −80° C. The morphologic analysis performed on a MVs suspension afterstaining with propidium iodide did not show the presence of apoptoticbodies.

RNase treatment. In selected experiments, MVs from HLSCs were treatedwith 1 U/ml RNase for 1 h at 37° C. The reaction was stopped by additionof 10 U/ml RNase inhibitor and MVs were washed by ultracentrifugation.

Cell Proliferation. In order to investigate whether MVs derived fromHLSCs exerted their anti-tumor activity on cell lines derived from avariety of tumors, MCF-7 breast adenocarcinoma cells and Kaposi'ssarcoma cells were seeded at 8,000 cells/well into 96-well plates inDMEM and RPMI, respectively, with different concentrations of MVs (2;10; 15; and 30 μg/ml and 30 μg/ml of RNase treated-MVs). DNA synthesiswas detected as incorporation of 5-bromo-2′-deoxy-uridine (BrdU) intothe cellular DNA after 48 hours of culture. Cells were then fixed with0.5 M ethanol/HCl and incubated with nuclease to digest the DNA. BrdUincorporated into the DNA was detected using an anti-BrdUperoxidase-conjugated mAb and visualized with a soluble chromogenicsubstrate. Optical density was measured with an ELISA reader at 405 nm.

Apoptotic assay. MCF-7 and KS cells were seeded at 8,000 cells/well into96-well plated in low glucose DMEM with 10% FCS and in the presence ofDoxorubicin (100 ng/ml) or different concentrations of MVs (2; 10; 15;and 30 μg/ml and 30 μg/ml of RNase treated MVs). Apoptosis was evaluatedusing the TUNEL assay.

2.7.2 Results MVs Derived from HLSCs Inhibit in Vitro Proliferation ofMCF-7 Cells and KS Cells

Incubation for 48 hours of MCF-7 breast adenocarcinoma cells andKaposi's sarcoma cells with 2, 10, 15 and 30 μg/ml of MVs (FIGS. 16 and17) derived from HLSC-6B cells significantly inhibits cell proliferationcompared to control cells incubated with vehicle alone. These resultsshow that the anti-tumour effects of tissue resident stem cells are notspecific against tumours originated from the same tissue. Moreover,incubation of MCF-7 breast adenocarcinoma cells and Kaposi's sarcomacells for 48 hours with 2, 10, 15 and 30 μg/ml of MVs (FIG. 18) derivedfrom HLSC-6B cells induced apoptosis, compared to control cellsincubated with vehicle alone, with effects which are similar to those ofdoxorubicine, a chemotherapeutic drug. This further confirms that theanti-tumour effects of tissue resident stem cells are not specificagainst tumours originated from the same tissue.

1-20. (canceled)
 21. A method for treating a tumor disease comprising administering to a patient in need thereof a therapeutically effective amount of microvesicles (MVs) derived from an adult stem cell and a therapeutically effective amount of at least, one of one or more cytotoxic agents and one or more cytostatic agents.
 22. The method according to claim 21, wherein the cytotoxic and cytostatic agents are selected from the group consisting of Paclitaxel, Lenalidomide, Pomalidomide, Eprirubicin, 5FU, Sunitinib, La-patinib, Canertinib, cyclophosphamide, doxorubicin, Lenalidomide/Dexamethasone, Pomalidomide/Dexamethasone, Carboplatin, Rapamycin, mitoxantron, oxaliplatin, docetaxel, vinorelbin, vincristine, and combinations thereof.
 23. The method according to claim 21, wherein the cytotoxic and cytostatic agents are doxorubicin or vincristine.
 24. The method according to claim 21, wherein the adult stem cell is a mesenchymal stem cell.
 25. The method according to claim 21, wherein the adult stem cell is a human mesenchymal stem cell derived from bone marrow (BM-MSC) or a human mesenchymal stem cell derived from decapsulated glomeruli (GI-MSC).
 26. The method according to claim 21, wherein the adult stem cell is a liver stem cell.
 27. The method according to claim 21, wherein the adult stem cell is a non-oval liver stem cell (HLSC).
 28. The method according to claim 21, wherein the tumour disease is selected from the group consisting of liver tumour, epithelial tumour, breast tumour, lung tumour, prostate tumour, gastric tumour, colon tumour, and ovarian tumour.
 29. The method according to claim 21, comprising administering the microvesicles in a dose of 0.1 to 200 micrograms/kg body weight of the patient.
 30. The method according to claim 21, comprising administering the microvesicles in a dose of 1-150 micrograms/kg body weight of the patient.
 31. The method according to claim 21, comprising administering the microvesicles in a dose of 3-120 micrograms/kg body weight in the patient.
 32. The method according to claim 21, wherein the microvesicles are administered through a local or a systemic route. 